A number of clinical situations could be benefited by a more rapid histological processing method, which reduces the number of steps between obtaining a fresh tissue specimen and rendering a diagnosis. For example, there is a need for rapid pathologic detection of residual cancer on the surface of tumor resection specimens, known as positive tumor margins, such that the surgical procedure can be revised in time to prevent cancer from being left behind in the patient. Tumor excision is a very common treatment in solid cancers, where the surgeon must remove all of the diseased tissue from the patient to minimize the risk of cancer recurrence. Complete surgical removal offers the best prognosis for many types of cancer, especially those that have not metastasized. In most cases, the goal of these surgeries or tumor resections is to achieve cancer-free (or negative) surgical margins, thereby lowering the risk of local tumor recurrence. Surgical margin status has been shown to be a powerful predictor of local recurrence in several cancers including breast, brain, oral, head and neck, hepatic, esophageal, colorectal, prostate, and cervical cancers, as well as in soft-tissue sarcomas located in the extremities, trunk, and head and neck regions.
Currently, there are very few intra-operative tools available to assist surgeons in tumor margin diagnosis to ensure complete removal of the tumor. Touch prep cytology and frozen section analysis are two intra-operative tools currently implemented in some clinics, but both require a trained pathologist and other resources. Intraoperative pathology for surgical tumor resections is expensive and time-consuming and consequently is only available at a handful of surgery centers in the US. Thus, there is a clear unmet clinical need for effective intraoperative tools that can quickly examine the microscopic properties of surgical margins. Optical imaging techniques are particularly attractive for this application as entire margins can be intra-operatively imaged non-destructively and, potentially, in situ.
Fluorescence microscopy can be combined with a suitable bio-marker to generate additional nuclear-specific contrast, thereby allowing for the quantitation of nuclear size and density. In conventional fluorescence microscopy, the entire field of view is uniformly illuminated with the excitation light. This creates a problem as fluorophores outside the plane of focus are also excited and emit photons, generating a significant amount of unwanted background fluorescence. This in turn significantly degrades contrast of features of interest in the focal plane. This is a common issue in wide-field fluorescence microscopy and several specialized techniques exist to reject background fluorescence, such as fluorescence confocal microscopy. While effective in rejecting background fluorescence, confocal microscopy requires specialized optics in the form of rapid scanning mirrors and adjustable pinhole collection, which increases the cost and decreases the speed, which hinders the intra-operative intraoperative clinical feasibility. In addition, the amount of photons collected at each beam scan position is limited, so the system requires a sensitive detector, such as a photomultiplier tube. Further, because of the small field of view, the beam position has to be raster scanned to be able to survey an area that is in on the order of square millimeter to square centimeter.